PROJECT SUMMARY Host defense peptides (HDPs), more than 2,700 of which are known, play an indispensable role as the first line of defense in multi-cellular organisms. Their unique combination of features, such as broad-spectrum efficacy against bacteria, vi- ruses, and tumors, immunomodulatory effects, and low incidence of bacterial drug resistance, has spurred efforts to inves- tigate their molecular mechanisms of action and develop them into a new generation of antimicrobial agents. However, using natural HDPs as prototypes to rationally engineer new anti-infective agents requires understanding the molecular features underlying their high potency, specificity, and mechanisms of action. Here, we employ host defense metallopep- tides from the piscidin family to investigate molecular models of HDP action in bacterial cell membranes. Piscidins are particularly active on a wide range of bacteria associated with dangerous human illnesses, including Vibrio species, MRSA, and C. difficile. They carry an amino-terminal copper and nickel binding (ATCUN) motif. We previously demon- strated that they coordinate Cu2+ in their biological environment and their antimicrobial potency is dependent on Cu2+ availability. We have also shown that the membrane activity of piscidin is correlated with secondary structure formation upon membrane binding and reorientation (tilting) in the bilayer at high peptide concentration. Intersecting our findings on piscidins is the discovery by one of our team members that several Vibrio species incorporate exogenous polyunsatu- rated FAs in their membranes, affecting bacterial virulence and the potency of antimicrobial agents. This proposal builds on our previous work on piscidin and human pathogens such as Vibrio and C. difficile, and seeks to establish a molecular basis for the two modalities used by piscidin to damage bacterial membranes and characterize the biological importance of this mechanistic plurality. Using comparative studies of wild-type and mutated piscidins in the metallated and unmetal- lated forms, we will perform experiments to discern the chemical and physical membrane effects that underlie their po- tency. Aims 1 and 2 of this proposal will use biophysical studies of mutants to identify functionally-important residues in piscidin, and solid-state NMR experiments to establish on a molecular level how these residues contribute to membrane activity. Aim 3 will investigate the biological activity of the HDPs, as needed to demonstrate the biological relevance of principles identified in Aims 1-2. This contribution is significant since it will explore the new paradigm that Cu2+-bound HDPs target physical and chemical vulnerabilities in the membranes of bacteria that can incorporate lipids from the host or natural environment to control virulence. Overall, these experiments will engage each year a total of four undergraduate and Ph.D. students at all levels of a project aimed at elucidating the mechanism of action of highly potent ATCUN-HDPs. The knowledge gained from this project could help design novel anti-infective therapeutics that leverage multiple modes of action to disrupt bacterial membranes.